This invention relates to the use of compounds as inhibitors of the fatty acid synthase FabH.
The pathway for the biosynthesis of saturated fatty acids is very similar in prokaryotes and eukaryotes. However, although the chemical reactions may not vary, the organization of the biosynthetic apparatus is very different. Vertebrates and yeasts possess type I fatty acid synthases (FASs) in which all of the enzymatic activities are encoded on one or two polypeptide chains, respectively. The acyl carrier protein (ACP) is an integral part of the complex. In contrast, in most bacterial and plant FASs (type II) each of the reactions are catalyzed by distinct monofunctional enzymes and the ACP is a discrete protein. Mycobacteria are unique in that they possess both type I and II FASs; the former is involved in basic fatty acid biosynthesis whereas the latter is involved in synthesis of complex cell envelope lipids such as mycolic acids. There therefore appears to be considerable potential for selective inhibition of the bacterial systems by broad-spectrum antibacterial agents (Jackowski, S. 1992. In Emerging Targets in Antibacterial and Antifungal Chemotherapy. Ed. J. Sutcliffe and N. Georgopapadakou. Chapman and Hall, New York; Jackowski, S. et al. (1989). J. Biol. Chem. 264, 7624-7629.)
The first step in the biosynthetic cycle is the condensation of malonyl-ACP with acetyl-CoA by FabH. In subsequent rounds malonyl-ACP is condensed with the growing-chain acyl-ACP (FabB and FabF, synthases I and II respectively). The second step in the elongation cycle is ketoester reduction by NADPH-dependent xcex2-ketoacyl-ACP reductase (FabG). Subsequent dehydration by xcex2-hydroxyacyl-ACP dehydrase (either FabA or FabZ) leads to trans-2-enoyl-ACP which is in turn converted to acyl-ACP by NADH-dependent enoyl-ACP reductase (FabI). Further rounds of this cycle, adding two carbon atoms per cycle, eventually lead to palmitoyl-ACP whereupon the cycle is stopped largely due to feedback inhibition of FabH and I by palmitoyl-ACP (Heath, et al., (1996), J. Biol. Chem. 271, 1833-1836). Fab H is therefore a major biosynthetic enzyme which is also a key regulatory point in the overall synthetic pathway (Heath, R. J. and Rock, C. O. 1996. J. Biol. Chem. 271, 1833-1836; Heath, R. J. and Rock, C. O. 1996. J. Biol. Chem. 271, 10996-11000).
The antibiotic thiolactomycin has broad-spectrum antibacterial activity both in vivo and in vitro and has been shown to specifically inhibit all three condensing enzymes. It is non-toxic and does not inhibit mammalian FASs (Hayashi, T. et al., 1984. J. Antibiotics 37, 1456-1461; Miyakawa, S. et al., 1982. J. Antibiotics 35, 411-419; Nawata, Y et al., 1989. Acta Cryst. C45, 978-979; Noto, T. et al., 1982. J. Antibiotics 35, 401-410; Oishi, H. et al., 1982. J. Antibiotics 35, 391-396. Similarly, cerulenin is a potent inhibitor of FabB and F and is bactericidal but is toxic to eukaryotes because it competes for the fatty-acyl binding site common to both FAS types (D""Agnolo, G. et al.,1973. Biochim. Biophys. Acta. 326, 155-166). Extensive work with these inhibitors has proved that these enzymes are essential for viability. Little work has been carried out in Gram-positive bacteria.
There is an unmet need for developing new classes of antibiotic compounds that are not subject to existing resistance mechanisms. No marketed antibiotics are targeted against fatty acid biosynthesis, therefore it is unlikely that novel antibiotics of this type would be rendered inactive by known antibiotic resistance mechanisms. Moreover, this is a potentially broad-spectrum target. Therefore, FabH inhibitors would serve to meet this unmet need.
This invention comprises cinnamate derivatives and pharmaceutical compositions containing these compounds and their use as FabH inhibitors that are useful as antibiotics for the treatment of Gram positive and Gram negative bacterial infections.
This invention further constitutes a method for treatment of a Gram negative or Gram positive bacterial infection in an animal, including humans, which comprises administering to an animal in need thereof, an effective amount of a compound of this invention.
The compounds of this invention are represented by Formula (I): 
wherein,
R1 is selected from the group consisting of H, C1-10 alkyl, C1-3 arylalkyl C1-3 heteroarylalkyl aryl, heteroaryl, C1-3alkyl-C3-6cycloalkyl, and C3-6cycloalkyl;
R2 is selected from the group consisting of H, O(CH2)m aryl, O(CH2)mheteroaryl, N(R5)(CH2)m heteroaryl, N(R5)(CH2)m heteroaryl, N(R6)COaryl, N(R6)COheteroaryl, N(R6)SO2aryl and N(R6)SO2heteroaryl wherein the aryl and hetroaryl moieties of R2 and R3 may be optionally substituted by one or more of CH3, CF3, OCF3, OH, OCH3, NH2, NHCH3, N(CH3)2, SCH3, SOCH3, SO2CH3, halogen, CO2H, CO2CH3) CONH2, CON(CH3)2, NHCOH, NHCOCH3, NHSO2CH3, methylenedioxy; provided that R2 is H when R3 is selected from the group consisting of O(CH2)maryl, O(CH2)mheteroaryl, N(R5)(CH2)m aryl, N(R5)(CH2)mheteroaryl, N(R6)COaryl, N(R6)COheteroaryl, N(R6)SO2aryl and N(R6)SO2heteroaryl;
R3 is selected from the group consisting of H, halogen, OCH3, CH3, O(CH2)maryl, O(CH2)mheteroaryl, N(R5)(CH2)m aryl, N(R5)(CH2)m heteroaryl, N(R6)COaryl, N(R6)COheteroaryl, N(R6)SO2aryl and N(R6)SO2heteroaryl; provided that R3 is selected from the group consisting of O(CH2)maryl, O(CH2)mheteroaryl, N(R5)(CH2)m aryl, N(R5)(CH2)m heteroaryl, N(R6)COaryl, N(R6)COheteroaryl, N(R6)SO2aryl and N(R6)SO2heteroaryl when R2 and R4 are H;
R4 is selected from the group consisting of H, halogen, OCH3, and CH3;
R5 is selected from the group consisting of H, C1-10 alkyl, C1-3 alkyl-aryl, C1-3 alkyl-heteroaryl CO(C1-8)alkyl, and COaryl and COheteroaryl;
R6 is selected from the group consisting of H, C1-10 alkyl,C1-3alkyl-aryl and C1-3alkyl-heteroaryl; and
m is an integer from 0-3;
or a pharmaceutically acceptable salt thereof.
Also included in the invention are pharmaceutically acceptable salt complexes.
As used herein, xe2x80x9cC1-10alkylxe2x80x9d or xe2x80x9calkylxe2x80x9d means both straight and branched chain of 1 to 10 carbon atoms, unless the chain length is otherwise limited, including, but not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl and the like. The alkyl may carry substituents such as hydroxy, carboxy, alkoxy, and the like.
The term xe2x80x9ccycloalkylxe2x80x9d is used herein to mean cyclic rings, preferably of 3 to 8 carbons, including but not limited to cyclopropyl, cyclopentyl, cyclohexyl, and the like.
The term xe2x80x9carylalkylxe2x80x9d or xe2x80x9cheteroarylalkylxe2x80x9d or xe2x80x9cheterocyclicalkylxe2x80x9d is used herein to mean C1-10 alkyl, as defined above, attached to an aryl, heteroaryl or heterocyclic moiety, as also defined herein, unless otherwise indicated.
As used herein,xe2x80x9carylxe2x80x9d means phenyl and naphthyl and substituted aryl such as hydroxy, carboxy, halo, alkoxy, methylenedioxy, etc.
As used herein, xe2x80x9cheteroarylxe2x80x9d means a 5-10 membered aromatic ring system in which one or more rings contain one or more heteroatoms selected from the group consisting of N, O or S, such as, but not limited, to pyrrole, pyrazole, furan, thiophene, quinoline, isoquinoline, quinazolinyl, pyridine, pyrimidine, oxazole, thiazole, thiadiazole, triazole, imidazole, benzotriazole, or benzimidazole. As used herein, preferred aryl substituents include halo, including chloro, fluoro, bromo and iodo, in any combination; C1-10 alkyl, C1-10 alkoxy, aryloxy, or heteroaryloxy.
The compounds of this invention may contain one or more asymmetric carbon atoms and may exist in racemic and optically active forms. All of these compounds and diastereomers are contemplated to be within the scope of the present invention.
Some of the compounds of this invention may be crystallised or recrystallised from solvents such as organic solvents. In such cases solvates may be formed. This invention includes within its scope stoichiometric solvates including hydrates as well as compounds containing variable amounts of water that may be produced by processes such as lyophilisation.
Since the antibiotic compounds of the invention are intended for use in pharmaceutical compositions it will readily be understood that they are each provided in substantially pure form, for example at least 60% pure, more suitably at least 75% pure and preferably at least 85%, especially at least 95% pure, particularly at least 98% pure (% are on a weight for weight basis). Impure preparations of the compounds may be used for preparing the more pure forms used in the pharmaceutical compositions; these less pure preparations of the compounds should contain at least 1%, more suitably at least 5% and preferably from 10 to 49% of a compound of the formula (I) or salt thereof.
Preferred compounds of the present invention are selected from the group consisting of:
(E)-2xe2x80x2-(6-Chloropiperonyl)-3-(4-phenoxyphenyl)cinnamic acid;
(E)-2xe2x80x2-(3-Bromophenyl)-3-(2,6-dichlorobenzyloxy)cinnamic acid;
(E)-3-(2,6-Dichlorobenzyloxy)-2xe2x80x2-[3-(4xe2x80x2-phenoxybiphenyl)]cinnamic acid;
(E)-2xe2x80x2-(3-Biphenyl)-3-(2,6-dichlorobenzyloxy)cinnamic acid;
(E)-2xe2x80x2-(6-Chloropiperonyl)-3-phenylcinnamic acid;
(E)-4-(2,6-Dichlorobenzyloxy)cinnamic acid;
(E)-2xe2x80x2-(6-Chloropiperonyl )-4-(2,6-dichlorophenoxy)cinnamic acid;
(Z)-2xe2x80x2-(6-Chloropiperonyl)-4-(2,6-dichlorophenoxy)cinnamic acid;
(Z)-2xe2x80x2-(6-Chloropiperonyl)-4-(3,5-dichlorophenoxy)cinnamic acid;
(E/Z)-2xe2x80x2-Benzyl4-(2,6-dichlorobenzyloxy)cinnamic acid;
(E)-2xe2x80x2-(6-Chloropiperonyl)-3,5-dichloro-4-(2,6-dichlorobenzyloxy)cinnamic acid;
(E)-3,5-Dichloro-4-(2,6-dichlorobenzyloxy)-2xe2x80x2-piperonylcinnamic acid;
(E/Z)-3-Chloro-4-(2,6-dichlorobenzyloxy)-5-methoxy-2xe2x80x2-piperonylcinnamic acid;
(E/Z)-4-(2,6-Dichlorobenzyloxy)-3-methoxy-2 xe2x80x2-piperonylcinnamic acid;
(E/Z)-4-(2,6-Dichlorobenzyloxy)-3,5-dimethy-2xe2x80x2-piperonylcinnamic acid;
(E)-2xe2x80x2-(6-Chloropiperonyl)-4-(3,5-dichloroanilino)cinnamic acid;
(E)-2xe2x80x2-(6-Chloropiperonyl)-4-(2,5-dichlorophenoxy)cinnamic acid;
(E)-2xe2x80x2-(6-Chloropiperonyl)-4-(4,6-dimethyl-pyrid-2-ylamino)cinnamic acid;
(E)-2xe2x80x2-(6-Chloropiperonyl)-4-(3,4-dichloroanilino)cinnamic acid;
(E)-2xe2x80x2-(6-Chloropiperonyl)-4-(3-cyanoanilino)cinnamic acid;
(E)-2xe2x80x2-(6-Chloropiperonyl)-4-(N-acetyl-3,4-dichloroanilino)cinnamic acid;
(E)-4-(2,6-Dichlorobenzyloxy)-2xe2x80x2-thiophenylcinnamic acid;
(E)-2xe2x80x2-(6-Chloropiperonyl)-4-(2,6-dichlorobenzylamino)cinnamic acid;
(E)-4-[Bis(2,6-dichlorobenzy)amino]-2xe2x80x2-(6-chloropiperonyl)cinnaric acid;
(E)-2xe2x80x2-(4-Aminobenzyl)-4-(2,6-dichlorobenzyloxy)cinnamic acid;
(E)-4-(2,6-Dichlorobenzyloxy)-2xe2x80x2-(4-methanesulfonaminobenzyl)cinnamic acid;
(E)-2xe2x80x2-Cyano4-(2,6-dichlorobenzyloxy)cinnamic acid;
(E)-4-(3,4-Dichlorobenzyloxy)-2xe2x80x2-(1-methyltetrazol-5-yl)cinnamic acid;
(E)-2xe2x80x2-(2-Cyanoethyl)-4-(2,6-dichlorobenzyloxy)cinnamic acid; and
(E)-4-(2,6-Dichlorobenzyloxy)-2xe2x80x2-(2-thienyl)cinnamic acid.
More preferred compound useful in the present invention are selected from the group consisting of:
(E)-2xe2x80x2-(6-Chloropiperonyl)-3-(2,6-dichlorobenzyloxy)cinnamic acid;
(E)-2xe2x80x2-(6-Chloropiperonyl)-4-(2,6-dichlorobenzyloxy)cinnamic acid;
(E)-2xe2x80x2-(6-Chloropiperonyl)-4-(3,5-dichlorophenoxy)cinnamic acid;
(E)-2xe2x80x2-(6-Chloropiperonyl)-4-(2,5-dichloroanilino)cinnamnic acid;
(E)-2xe2x80x2-(6-Chloropiperonyl)-4-(3,5-dichlorophenyl)cinnamic acid;
(E)-2xe2x80x2-(6-Chloropiperonyl)-4-(2-chloro-5-hydroxybenzyloxy)cinnamic acid;
(E)-2xe2x80x2-(Phenyl)-4-(2,6-dichlorobenzyloxy)cinnamic acid;
(E)-2xe2x80x2-(6-Chloropiperonyl)-4-(3-amino-2,6-dichlorobenzyloxy)cinnamic acid; and
(E)-4-(2,6-Dichlorobenzyloxy) 2xe2x80x2-(3-thienyl)cinnamic acid.
Compounds of Formula (I) wherein R2 or R3 is benzyloxy are prepared by the methods described in Scheme 1. 
a) NaH, DMF; b) NaH, DMF; c) KOH, EtOH; d) acetic acid, piperidine, benzene; e) NaOH, CH3OH, THF, H2O
Hydroxy benzaldehyde 1-Scheme-1, and a benzyl halide (such as 2,6-dichlorobenzyl bromide) are treated with a base (such as sodium hydride) in a solvent (such as DMF) and stirred (6 hours to 30 hours, preferably 16 hours) to yield 3-Scheme-1. Diethyl malonate 5-Scheme-1 is alkylated with a benzyl halide (such as 6-chloropiperonyl chloride 4-Scheme-1) and a base (such as sodium hydride) in a solvent (such as DMF) then stirred (1 hours to 10 hours, preferably 3 hours). The resulting diester is mono-saponified using a base (such as potassium hydroxide) in a solvent (such as ethanol) and stirred (4 hours to 30 hours, preferably 18 hours) to yield 6-Scheme-1. Knoevenagel condensation of 3-Scheme-1 with 6-Scheme-1 in a solvent (such as benzene) with catalysts (such as piperidine and acetic acid) at reflux temperature with azeotropic water removal provides the ester 7-Scheme-1. Saponification of 7-Scheme-1 with a base (such as potassium hydroxide) in a solvent (such as ethanol and tetrahydrofuran) provides 8-Scheme-1.
Compounds of Formula (I) wherein R2 is phenoxy or aninilo are prepared by the methods described in Scheme 2. 
a) acetic acid, piperidine, benzene; b) Cu(Oac)2, TEA, 4A sieves, CH2Cl2; c) KOH, EtOH
A Knoevenagel condensation of 1-Scheme-2 with 2-Scheme-2 (prepared by treating the arylboronic acid with ethylene glycol with concurrent water removal) in a solvent (such as benzene) with catalysts (such as piperidine and acetic acid) at reflux temperature with azeotropic water removal provides the boronic acid 3-Scheme-2. This is coupled with a substituted phenol or aniline (such as 3,5-dichloroaniline) using copper salts (such as Cu(II) acetate) and a base (such as TEA) with molecular sieves in a solvent (such as methylene chloride) to provide 4-Scheme-2. Saponification of 4-Scheme-2 with a base (such as potassium hydroxide) in a solvent (such as ethanol) provides 5-Scheme-2.
Compounds of Formula (I) wherein R2 is phenyl are prepared by the methods described in Scheme 3. 
a) ArX, (Ph3P)4Pd(0), Na2CO3, toluene, H2O; b) KOSi(CH3)3, THF
A Suzuki coupling of 1-Scheme-3 with an aryl halide (such as 3,5-dichloroiodobenzene) in the presence of a palladium catalyst [such as tetrakis(triphenylphosphine)palladium(0)] and a base (such as sodium carbonate) in a solvent (such as toluene and water) provides the ethyl ester of 2-Scheme-3. Saponification of the ethyl ester of 2-Scheme-3 with a base (such as potassium trimethylsilanolate) in a solvent (such as THF) provides 2-Scheme-3. 
a) acetic anhydride, TEA
A condensation of an arylacetic acid and a 4-benzyloxybenzaldehyde in acetic anhydride with triethyl amine at high temperature provides the 2xe2x80x2-arylcinnamic acids 3-scheme-4.